ObjectiveHerein we propose a combined action of collagen type I (CA) or synthetic collagen-like-peptide functionalized with the cell adhesive RGD motif (PEG-CLP-RGD) hydrogels and selected growth factors to promote chondrogenic differentiation of human muscle-derived stem cells (hMDSCs) under normal and reduced oxygen conditions.MethodshMDSCs were set for differentiation towards chondrogenic lineage using BMP-7 and TGF-β3. Cells were seeded onto hydrogels loaded with growth factors (75ng/scaffold) and cultured for 28 days under normal (21%) and severe hypoxic (1%) conditions. Chondrogenesis was evaluated by monitoring collagen type II and GAG deposition, and quantification of ACAN expression by RT-PCR.ResultsSustained release of TGFβ3 from the hydrogels was observed, 8.7 ± 0.5% of the initially loaded amount diffused out after 24 h from both substrates. For the BMP-7 growth factor, 14.8 ± 0.3% and 18.2 ± 0.6% of the initially loaded amount diffused out after 24 h from CA and CLP-RGD, respectively. The key findings of this study are: i) the self-supporting hydrogels themselves can stimulate hMDSC chondrogenesis by inducing gene expression of cartilage-specific proteoglycan aggrecan and ECM production; ii) the effect of dual BMP-7 and TGF-β3 loading was more pronounced on CA hydrogel under normal oxygen conditions; iii) dual loading on PEG-CLP-RGD hydrogels did not have the synergistic effect, TGF-β3 was more effective under both oxygen conditions; iv) BMP-7 can improve chondrogenic effect of TGF-β3 on CA scaffolds, and hydrogels loaded with both growth factors can induce cartilage formation in hMDSC cultures.ConclusionOur results support the potential strategy of combining implantable hydrogels functionalized with differentiation factors toward improving cartilaginous repair.

Background and Objectives: To date, the therapeutic potential of skeletal muscle-derived stem/progenitor cells (MDSPCs) for acute kidney injury (AKI) has only been evaluated by our research group. We aimed to compare MDSPCs with bone marrow mesenchymal stem cells (BM-MSCs) and evaluate their feasibility for the treatment of AKI. Materials and Methods: Rats were randomly assigned to four study groups: control, GM (gentamicin) group, GM+MDSPCs, and GM+BM-MSCs. AKI was induced by gentamicin (80 mg/kg/day; i.p.) for 7 consecutive days. MDSPCs and BM-MSCs were injected 24 h after the last gentamicin injection. Kidney parameters were determined on days 0, 8, 14, 21, and 35. Results: MDSPCs and BM-MSCs accelerated functional kidney recovery, as reflected by significantly lower serum creatinine levels and renal injury score, higher urinary creatinine and creatinine clearance levels (p < 0.05), lower TUNEL-positive cell number, and decreased KIM-1 and NGAL secretion in comparison to the non-treated AKI group. There was no significant difference in any parameters between the MDSPCs and BM-MSCs groups (p > 0.05). Conclusions: MDSPCs and BM-MSCs can migrate and incorporate into injured renal tissue, resulting in a beneficial impact on functional and morphological kidney recovery, which is likely mediated by the secretion of paracrine factors and an anti-apoptotic effect. MDSPCs were found to be non-inferior to BM-MSCs and therefore can be considered as a potential candidate strategy for the treatment of AKI.

Purpose This study aimed to conduct arthroscopic evaluation of cartilage electromechanical properties and establish their correlation with International Cartilage Repair Society (ICRS) grading scores. Methods In 18 patients, quantitative parameter (QP) measurements were taken on the weight-bearing surface of the medial femoral condyle. Adjacently, the same site was graded using ICRS scores (0-4). Electromechanical QPs for ICRS grades 0 to 3 were obtained during arthroscopy, while complete grade 4 injuries were assessed using femur cartilage-bone blocks from knee arthroplasty. The QP values for ICRS grades 0 to 2 were compared with grades 3 and 4 using Welch t test. The corresponding QP values were assigned to ICRS grades 0 to 4 and compared using Welch ANOVA (analysis of variance). Pearson’s coefficient evaluated QP-ICRS grade relationship. Results Healthy grade 0 cartilage displayed a mean QP value of 10.5 (±2.8 SD, n = 4). The ICRS grade 1 and grade 2 injuries were associated with QP values of 12 (±0.7, n = 2) and 13.25 (±1.77, n = 2), respectively. The grade 3 defects had QP values of 20.43 (±4.84, n = 4), whereas complete grade 4 defects showed electromechanical values of 30.17 (±2.19, n = 6). Significant differences in QP values were observed between ICRS grades 0 to 2 (mean QP 11.56 ± 2.3, n = 8) and grades 3 and 4 (26.27 ± 6, n = 10; P < 0.0001). Pearson’s correlation coefficient of 0.9 indicated a strong association between higher ICRS cartilage injury grades and elevated QP values ( P < 0.0001). Conclusion Arthroscopic electromechanical QP assessment robustly correlates with ICRS scores. The QP values for ICRS grades 0 to 2 are significantly lower, compared with grades 3 and 4.

Herein we address, hyaline cartilage regeneration issue by engineering a synthetic biocompatible hydrogel scaffold capable to promote chondrogenic differentiation. In this study, the chemically crosslinked hydrogels consisting of synthetic peptides that have the collagen-like sequence Cys-Gly-(Pro-Lys-Gly)4 (Pro-Hyp-Gly)4 (Asp-Hyp-Gly)4- conjugated with RGD sequence (CLP-RGD) and crosslinked hydrogels of type I collagen (CA) were used. For cartilage formation, we used human skeletal muscle-derived stem/progenitor cells (hMDSPCs) set for differentiation towards a chondrogenic lineage by BMP-7 and TGF-ß3 growth factors. Initially 150, 100 and 75 ng of BMP-7and TGF-ß3 growth factors were inserted in each scaffold and amount of growth factors diffusing out of the scaffolds was observed by ELISA assays. In vitro experiments were performed by seeding hMDSPCs onto hydrogels loaded with growth factors (75ng/scaffold) and cultured for 28 days. Cartilage formation was monitored by ELISA and RT-PCR assays. All experiments were performed in triplicates or quadruplicates. Growth factors incorporation strategy allowed a sustained release of TGF-ß3 growth factor, 6.00.3% of the initially loaded amount diffused out after 4 h and 2.70.5% already at the second time point (24h) from CA and CLP-RGD substrates. For the BMP-7 growth factor, 13.12.3% and 15.751.6% of the initially loaded amount diffused out after 4 h, 1.70.2% and 2.450.3% at the second time point (24 h) from CA and CLP-RGD respectively. In vitro experiments shown that scaffolds with immobilized growth factors resulted in higher collagen type II accumulation when compared to the scaffolds alone. The gene expression on CLP-RGD hydrogels with growth factors has shown lower collagen type I expression and higher aggrecan expression compared to day 0. However, we also report increased collagen X gene expression on CA hydrogels (with growth factors). Our results support the potential of the strategy of combining hydrogels functionalized with differentiation factors toward improving cartilage repair.

An advanced therapy construct for regeneration of articular cartilage is reported which was fabricated by cryo-electrospinning as 3D multi-layered poly[e]caprolactone matrix harbouring chondrogenic growth factor TGF beta-3. The layers have been designed to favour the cartilage regeneration (chondral layer) and bone tissue formation (subchondral layer) by additives of regenerated cellulose and hydroxyapatite, respectively. The overall porosity of the chondral layer was 90.7% and the subchondral layer - 94.4% with a pore diameter ranges of 50-300 mu m and 75-400 mu m respectively but maintaining a low fibre diameter of 9.3 +/- 4.1 mu m and 9.1 +/- 2.1 mu m. The hydrophilicity was enhanced and functional groups essential for growth factor incorporation were introduced by surface ozone treatment. The scaffold was characterised by SEM, mu CT, FTIR, WCA, XRD, TGA, and DSC techniques. The processing of scaffold by chemical modifications resulted in the increase of biodegradability of the scaffold, as indicated by the decrease of the crystallinity, thermal stability, melting peak, and hydrophobicity, but maintaining its sufficient mechanical properties (Young's modulus equalled to 1.8 +/- 0.1 MPa for chondral layer and 1.1 +/- 0.2 MPa for subchondral). The release kinetics of TGF beta-3 followed the Korsmeyer-Peppas model, reaching 39.3% after 72 h, and the estimated 50% release after 155 h.

Polycaprolactone (PCL) is a non-cytotoxic, completely biodegradable biomaterial, ideal for cartilage tissue engineering. Despite drawbacks such as low hydrophilicity and lack of functional groups necessary for incorporating growth factors, it provides a proper environment for different cells, including stem cells. In our study, we aimed to improve properties of scaffolds for better cell adherence and cartilage regeneration. Thus, electrospun PCL–scaffolds were functionalized with ozone and loaded with TGF-β3. Together, human-muscle-derived stem cells (hMDSCs) were isolated and assessed for their phenotype and potential to differentiate into specific lineages. Then, hMDSCs were seeded on ozonated (O) and non-ozonated (“naïve” (NO)) scaffolds with or without protein and submitted for in vitro and in vivo experiments. In vitro studies showed that hMDSC and control cells (human chondrocyte) could be tracked for at least 14 days. We observed better proliferation of hMDSCs in O scaffolds compared to NO scaffolds from day 7 to day 28. Protein analysis revealed slightly higher expression of type II collagen (Coll2) on O scaffolds compared to NO on days 21 and 28. We detected more pronounced formation of glycosaminoglycans in the O scaffolds containing TGF-β3 and hMDSC compared to NO and scaffolds without TGF-β3 in in vivo animal experiments. Coll2-positive extracellular matrix was observed within O and NO scaffolds containing TGF-β3 and hMDSC for up to 8 weeks after implantation. These findings suggest that ozone-treated, TGF-β3-loaded scaffold with hMDSC is a promising tool in neocartilage formation.

Polycaprolactone (PCL) has recently received significant attention due to its mechanical strength, low immunogenicity, elasticity, and biodegradability. Therefore, it is perfectly suitable for cartilage tissue engineering. PCL is relatively hydrophobic in nature, so its hydrophilicity needs to be enhanced before its use in scaffolding. In our study, first, we aimed to improve the hydrophilicity properties after the network of the bilayer scaffold was formed by electrospinning. Electrospun bilayer PCL scaffolds were treated with ozone and further loaded with transforming growth factor-beta 3 (TGFβ3). In vitro studies were performed to determine the rabbit muscle-derived stem cells' (rMDSCs) potential to differentiate into chondrocytes after the cells were seeded onto the scaffolds. Statistically significant results indicated that ozonated (O) scaffolds create a better environment for rMDSCs because collagen-II (Coll2) concentrations at day 21 were higher than non-ozonated (NO) scaffolds. In in vivo studies, we aimed to determine the cartilage regeneration outcomes by macroscopical and microscopical/histological evaluations at 3- and 6-month time-points. The Oswestry Arthroscopy Score (OAS) was the highest at both mentioned time-points using the scaffold loaded with TGFβ3 and rMDSCs. Evaluation of cartilage electromechanical quantitative parameters (QPs) showed significantly better results in cell-treated scaffolds at both 3 and 6 months. Safranin O staining indicated similar results as in macroscopical evaluations-cell-treated scaffolds revealed greater staining with safranin, although an empty defect also showed better results than non-cell-treated scaffolds. The scaffold with chondrocytes represented the best score when the scaffolds were evaluated with the Mankin histological grading scale. However, as in previous in vivo evaluations, cell-treated scaffolds showed better results than non-cell-treated scaffolds. In conclusion, we have investigated that an ozone-treated scaffold containing TGFβ3 with rMDSC is a proper combination and could be a promising scaffold for cartilage regeneration.

Ozonation has been proved as a viable surface modification technique providing certain properties to the scaffolds that are essential in tissue engineering. However, the ozone (O3) treatment of PCL scaffolds in aqueous environments has not yet been presented. O3 treatment performed in aqueous environments is more effective compared with traditional, executed in ambient air treatment due to more abundant production of hydroxyl radicals (•OH) within the O3 reaction with water molecules. During interaction with •OH, the scaffold acquires functional groups which improve wettability properties and encapsulate growth factors. In this study, a poly(ε)caprolactone (PCL) scaffold was fabricated using solution electrospinning and was subsequently ozonated in a water reactor. The O3 treatment resulted in the expected occurrence of oxygen-containing functional groups, which improved scaffold wettability by almost 27% and enhanced cell proliferation for up to 14 days. The PCL scaffold was able to withhold 120 min of O3 treatment, maintaining fibrous morphology and mechanical properties.

Objective To determine the applicability of a minimally invasive diagnostic device to evaluate the quality of articular cartilage following autologous (OAT) and allogeneic (OCA) osteochondral graft transplantation in goat model. Design OAT grafts were harvested from lateral femoral condyles (LFCs) and transplanted into osteochondral defects created in medial femoral condyles (MFCs) of contralateral knees. OCA grafts were transplanted into MFC condyles afterin vitrostorage. Autologous platelet-rich plasma (PRP) was administered intraarticularly after the surgery and at 1 and 2 months postoperatively. OAT and OCA grafts were evaluated macroscopically (Oswestry arthroscopy score [OAS]), electromechanically (quantitative parameter, QP), and histologically (O'Driscoll score, safranin O staining intensity) at 3 and 6 months after transplantation. Results were compared with preoperative graft evaluation. Results Transplanted cartilage deteriorated within 6 months in all groups. Cartilage quality was better retained in OAT group compared with a decline in OCA group. QP and OAS scores were comparable in OAT and OCA groups at 3 months, but superior in OAT group at 6 months, according to all the methods applied. PRP injections significantly improved QP and OAS score at 6 months compared with 3 months in OAT group. QP moderately correlated with OAS, O'Driscoll score, and safranin O staining intensity. Conclusions Grafts did not retain preoperative quality parameters at 6 months follow-up; however, OAT were superior to OCA grafts. PRP may have a beneficial effect on macroscopic and electromechanical properties of cartilage; however, histological improvement is yet to be proved. Electromechanical diagnostic device enables reliable assessment of transplanted cartilage.

Direct laser writing 3D lithography in pre-polymers was employed to microstructure custom 3D silicon-zirconium hybrid organic-inorganic polymer SZ2080 scaffolds (HOI) of varying morphology for cartilage repair in a preclinical xenogeneic model. Scaffolds were fabricated to contain tetragonal and hexagonal pores, followed by pore scaling of 1.5 and 2 times. HOI scaffolds were seeded with human chondrocytes (cells) and biocompatibility was analyzed in vitro. Tissue engineered cartilage (TEC) potency, efficacy and shelf-life in vitro was assessed by morphological, biomechanical, metabolic activity, cell count, ELISA and PCR analysis. Optimal HOI scaffold was implanted in a long-term preclinical osteochondral defect of immunodeficient rat model and analyzed for the translated efficacy in experimental groups. Collagen scaffold was a positive comparator for in vitro and in vivo studies. Treatment efficacy was evaluated after 3 months using standardized macroscopical and histological scores. Biocompatibility was superior in tetragon-pored scaffold (HOI-T) compared to hexagon-pored HOI in vitro. Cartilage tissue formation in HOI with tetragonal pores scaled 1.5 times was comparable to HOI-T at least for up to 7 days in vitro. HOI-T with and without cells improved cartilage repair and were comparable to collagen scaffold in vivo at 3-months follow-up.

Polymer-based scaffolds with immobilised hydroxyapatite particles are among the most extensively studied materials for bone tissue regeneration. In this study, cellulose-based scaffolds with immobilised nanohydroxyapatite and microhydroxyapatite particles were prepared and analysed by micro-computed tomography. The scaffolds contained non-symmetrical interconnected pores. The porosity of the cellulose/nanohydroxyapatite and the cellulose/microhydroxyapatite scaffolds was 72% and 66%, respectively. The cytotoxicity of the cellulose-based scaffolds to hepatocytes and skeletal muscle tissue was evaluated. The results showed that the nanohydroxyapatite and the cellulose scaffolds containing nanoparticles reduced liver cell viability and increased the release of lactate dehydrogenase and aldolase. Moreover, the scaffolds containing nanohydroxyapatite particles caused cell plasma membrane damage that was manifested by significantly reduced insulin-stimulated glycogen synthesis in liver cells and glucose uptake by skeletal muscle cells. Controversially, microhydroxyapatite and the cellulose/microhydroxyapatite scaffolds had no deteriorating effect on cell survival, plasma membrane damage and glucose metabolism.

Arthroscopic surgery has grown rapidly in recent decades. Despite accurately diagnosed clinical cases, the previous pain is retained in some patients after the operation, even though no visible chondral lesions are found during the procedure. A minimally invasive arthroscopic method of measuring articular cartilage electromechanical properties enables rapid and reliable intraoperative articular cartilage quality evaluation.

Skeletal muscle-derived stem/progenitor cells (MDSPCs) have been thoroughly investigated and already used in preclinical studies. However, therapeutic potential of MDSPCs isolated using preplate isolation technique for acute kidney injury (AKI) has not been evaluated. We aimed to characterize rat MDSPCs, compare them with bone marrow mesenchymal stem cells (BM-MSCs), and evaluate the feasibility of MDSPCs therapy for gentamicin-induced AKI in rats. We have isolated and characterized rat MDSPCs and BM-MSCs. Characteristics of rat BM-MSCs and MDSPCs were assessed by population doubling time, flow cytometry, immunofluorescence staining, RT-PCR, and multipotent differentiation capacity. Gentamicin-induced AKI model in rat was used to examine MDSPCs therapeutic effect. Physiological and histological kidney parameters were determined. MDSPCs exhibited similar immunophenotype, stem cell gene expression, and multilineage differentiation capacities as BM-MSCs, but they demonstrated higher proliferation rate. Single intravenous MDSPCs injection accelerated functional and morphological kidney recovery, as reflected by significantly lower serum creatinine levels, renal injury score, higher urinary creatinine, and GFR levels. PKH-26-labeled MDSPCs were identified within renal cortex 1 and 2 weeks after cell administration, indicating MDSPCs capacity to migrate and populate renal tissue. In conclusion, MDSPCs are capable of mediating functional and histological kidney recovery and can be considered as potential strategy for AKI treatment.

Purpose: Evaluate autologous matrix-induced chondrogenesis (AMIC) treatment outcomes for characterised cartilage lesions. Evaluate untreated defects impact on clinical outcome. Methods: Fifteen patients (median age 30.7 years) actively engaged in recreational sports activities with symptomatic cartilage lesions of the knee were studied. Defects were characterised intraoperatively by count, size, grade and the area that received treatment. Lesions classified as grade III and IV by International Cartilage Repair Society (ICRS) were treated with AMIC or AMIC in conjunction with other cartilage repair techniques, whereas grade I and II lesions were left untreated. Two treatment exceptions were made. Patients from the Total group and four separate subgroups (Single; Multiple; Treated; Partly treated) were evaluated by ICRS and Tegner scores at the median follow-up of 4.5 years. Results: Total group patients were diagnosed with twenty-eight articular cartilage defects (1.9 per patient). The average defect size was 5.3±2.2 cm2, of which 57% were classified as grade III and IV lesions. Patients from the Multiple subgroup had significantly greater diagnosed (6.9±2.1 cm2, P=.012) and untreated (2.6±2.6 cm2, P=.038) lesions areas, when compared to the Single subgroup. Partly treated subgroup had a significantly larger area of untreated defect (3.6±2.3 cm2, P=.025), when compared to the Treated subgroup. The average ICRS preoperative values of the Total group and individual subgroups improved significantly at the follow-up. Significantly more patients retained their previous activity levels (P=.026) and had a higher incremental ICRS score (P=.014) in the Single subgroup when compared to the Multiple subgroup. Total defect size diagnosed negatively correlated to ICRS incremental score (r=-.624, P=.023) and had a tendency to postoperative ICRS score correlation (r=-.545, P=.054). Conclusions: AMIC technique can be effectively applied for patients with single and multiple cartilage lesions of the knee albeit clinical outcome is superior in patients with a single defect per knee. Significantly higher return rate to previous physical activity levels was noted after every cartilage defect is treated. Total diagnosed defect area negatively correlate to ICRS score increment at the follow-up.Tabled 1Defect area characteristics, cm2SingleMultipleP ValueTreatedPartly treatedP ValueTotal defect area diagnosed4.0±1.316.86±2.12.0174.7±2.06.6±2.3.158Total defect area treated4.0±1.314.29±2.36.824.7±2.03.0±0.0.075Total defect area left untreated0.02.57±2.57.0230.03.6±2.3.003 Open table in a new tab Tabled 1Clinical outcomesGroupICRS scorepreoperativeICRS score postoperativeICRS score improvementReturnedDid not returnTotal55.54±5.3586.54±6.4531±5.4211 (73%)4 (27%)Single55.29±6.0789.71±4.1934.43±4.798 (100%)0Multiple55.83±4.9282.83±6.9427±2.683 (42.9%)4 (57.1%)Treated54.67±5.4587.11±6.3132.44±5.778 (80%)2 (20%)Partly treated57.5±5.2685.25±7.5427.75±2.873 (60%)2 (40%) Open table in a new tab View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

This study investigated the role of cyclooxygenase-2 (COX-2) expression by donor and host cells in muscle-derived stem cell (MDSC)-mediated bone regeneration utilizing a critical size calvarial defect model. We found that BMP4/green fluorescent protein (GFP)-transduced MDSCs formed significantly less bone in COX-2 knock-out (Cox-2KO) than in COX-2 wild-type (WT) mice. BMP4/GFP-transduced Cox-2KO MDSCs also formed significantly less bone than transduced WT MDSCs when transplanted into calvarial defects created in CD-1 nude mice. The impaired bone regeneration in the Cox-2KO MDSCBMP4/GFP group is associated with downregulation of BMP4-pSMAD1/5 signaling, decreased osteogenic differentiation and lowered proliferation capacity after transplantation, compared with WT MDSCBMP4/GFP cells. The Cox-2KO MDSCBMP4/GFP group demonstrated a reduction in cell survival and direct osteogenic differentiation in vitro These effects were mediated in part by the downregulation of Igf1 and Igf2. In addition, the Cox-2KO MDSCBMP4/GFP cells recruited fewer macrophages than the WT MDSC/BMP4/GFP cells in the early phase after injury. We concluded that the bone regeneration capacity of Cox-2KO MDSCs was impaired because of a reduction in cell proliferation and survival capacities, reduction in osteogenic differentiation and a decrease in the ability of the cells to recruit host cells to the injury site.